BackgroundInsulin glargine (Lantus®) is a long-acting basal insulin analog that demonstrates effective day-long glycemic control and a lower incidence of hypoglycemia than NPH insulin. After subcutaneous injection insulin glargine is partly converted into the two main metabolites M1 ([GlyA21]insulin) and M2 ([GlyA21,des-ThrB30]insulin). The aim of this study was to characterize the glargine metabolites in vitro with regard to their insulin receptor (IR) and IGF-1 receptor (IGF1R) binding and signaling properties as well as their metabolic and mitogenic activities.MethodsThe affinity of human insulin, insulin glargine and its metabolites to the IR isoforms A and B or IGF1R was analyzed in a competitive binding assay using SPA technology. Receptor autophosphorylation activities were studied via In-Cell Western in CHO and MEF cells overexpressing human IR-A and IR-B or IGF1R, respectively. The metabolic response of the insulins was studied as stimulation of lipid synthesis using primary rat adipocytes. Thymidine incorporation in Saos-2 cells was used to characterize the mitogenic activity.ConclusionsThe binding of insulin glargine and its metabolites M1 and M2 to the IR were similar and correlated well with their corresponding autophosphorylation and metabolic activities in vitro. No differences were found towards the two IR isoforms A or B. Insulin glargine showed a higher affinity for IGF1R than insulin, resulting in a lower EC50 value for autophosphorylation of the receptor and a more potent stimulation of thymidine incorporation in Saos-2 cells. In contrast, the metabolites M1 and M2 were significantly less active in binding to and activation of the IGF1R and their mitogenicity in Saos-2 cells was equal to human insulin. These findings strongly support the idea that insulin glargine metabolites contribute with the same potency as insulin glargine to blood glucose control but lead to significantly reduced growth-promoting activity.

Background In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R), present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids. Methodology To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET) assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP 3 ) production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP 3 production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells. Results Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP 3 production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP 3 production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin. Conclusion Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in MCF-7 cells.

The potency of oxalyl amino acid derivatives as inhibitors of prolyl 4-hydroxylase was studied in vitro, in isolated microsomes and in chicken embryonic-tissue culture. These compounds represent structural analogues of 2-oxoglutarate in which the -CH2- moiety at C-3 is replaced by -NH-, with or without further structural modifications. The most efficient inhibitor of purified prolyl 4-hydroxylase was oxalylglycine. Its mode of inhibition was competitive with respect to 2-oxoglutarate. The Ki value varied between 1.9 and 7.8 microM, depending on the variable substrate used. Oxalylalanine inhibited purified enzyme with a Ki of 40 microM. Other oxalyl amino acid derivatives showed little inhibitory activity. In microsomes isolated from embryonic chicken bone, oxalylglycine and oxalylalanine inhibited prolyl hydroxylation with IC50 values of 23 and 120 microM respectively. Dimethyloxalylglycine was not an inhibitor of purified prolyl 4-hydroxylase and only weakly active in the microsomal system, but efficiently suppressed hydroxyproline synthesis in embryonic chicken calvaria and lung. The data suggest that dimethyloxalyl amino acids are converted into active inhibitors in intact cells, most likely in the cytoplasmic compartment.

Insulin-resistant skeletal muscle of ZDF rats is characterised by a specific gene expression profile with increased levels of Scd1. An insulin-resistant phenotype similar to that obtained by treatment with palmitate and high glucose can be induced in vitro by overexpression of SCD1 in muscle cells. This supports the hypothesis that elevated SCD1 expression is a possible cause of insulin resistance and type 2 diabetes.

Chronic damage to the liver by a variety of causes frequently results in fibrogenesis, the increased deposition of extracellular matrix (ECM) proteins. The main component of the newly formed ECM is collagen.